Magnetic resonance imaging is a noninvasive imaging modality in medical diagnosis with excellent soft tissue discrimination and high spatial resolution that outstrips conventional imaging techniques such as computed tomography (CT) and positron emission tomography (PET). With the unveiled human genome and advances in molecular biology, MRI has a far more wide ranging scope than it was originally envisaged. It penetrates into a variety of perspectives in the medical field such as the thornostic perspective, in the chemical engineering field, such as in the characterization of fluid flows and the visualization of structure-hydrodynamics relationships, and into pharmaceutical research, such as in the use in drug development that relates observation with the physiological mechanism. MRI constructs images by making use of the inherent natural abundance and the nature of the proton spins of the water molecules in human bodies. A superconducting magnet with homogenous magnetic field and radiofrequency pulses are the hardware, and the pharmaceutical contrast agents are the software. The use of contrast agents can improve the intrinsic poor sensitivity, especially in applications such as the targeting of specific cells or tissues at low concentration. Approximately 40% of MRI tests are contrast-enhanced annually, and this percentage is increasing, particularly with the urgent need for better molecular targeting contrast agents.
The biophysics of MRI contrast agents is the alternation of the relaxation rate, which is governed by paramagnetism. In addition to the proton density, two major mechanisms contribute to the degree of contrast enhancement: the longitudinal (T1) and transverse relaxation (T2). Relaxivity is a measure of the efficacy of the paramagnetic complexes in shortening the time for the relaxation processes at 1 mM concentration: a large value usually reflects a better in vivo performance. The small molecular gadolinium-based contrast agents are efficient T1-agents that shorten the relaxation time and hence increase the relaxation rate. The observed relaxivity is composed of the innersphere relaxivity R1pIS, the outersphere relaxivity R1pOS, and the relaxation rate of the solvent in the absence of the paramagnetic complex, as shown in equation 1:R1obs=R1pIS+R1pOS+R1W  Equation 1
Various parameters define the effects of the contrast agents on the relaxation mechanisms, such as the electronic correlation time τv, the reorientational correlation time τR, the residence lifetime τm, etc. These parameters are interdependent. A paramagnetic metal centre such as iron, manganese, and gadolinium, determines the electronic relaxation time; gadolinium is preferred and widely used because of its seven unpaired electrons and the symmetric S-state that provide a large magnetic moment and a slow electronic relaxation rate. The control of the image contrast mainly depends on the longitudinal innersphere relaxation, which is expressed by Solomon-Bloembergen equations as equation 2-5 (Solomon, Phys. Rev., 1955, 99, 559) (Bloembergen, J. Chem. Phys., 1957, 27, 572) (Solomon and Bloembergen, J. Chem. Phys., 1956, 25, 261).
                                          1                          T              1                                =                                    R                              1                ⁢                                                                  ⁢                p                            IS                        =                                                            [                  M                  ]                                ⁢                q                                            55.6                ⁢                                  (                                                            T                                              1                        ⁢                                                                                                  ⁢                        M                                                              +                                          τ                      m                                                        )                                                                    ,                            Equation        ⁢                                  ⁢        2                                                      1                          T                              1                ⁢                M                                              =                                    1                              T                1                DD                                      +                          1                              T                1                SC                                                    ,                            Equation        ⁢                                  ⁢        3                                                      1                          T              1              DD                                =                                                    2                15                            ⁡                              [                                                                            γ                      I                      2                                        ⁢                                          g                      2                                        ⁢                                          μ                      B                      2                                        ⁢                                          S                      ⁡                                              (                                                  S                          +                          1                                                )                                                                                                  r                    GdH                    6                                                  ]                                      ⁢                                          (                                                      μ                    0                                                        4                    ⁢                                                                                  ⁢                    π                                                  )                            2                        ⁢                          (                                                                    7                    ⁢                                          τ                                              c                        ⁢                                                                                                  ⁢                        2                                                                                                  1                    +                                                                  ω                        s                        2                                            ⁢                                              τ                                                  c                          ⁢                                                                                                          ⁢                          2                                                2                                                                                            +                                                      3                    ⁢                                          τ                                              c                        ⁢                                                                                                  ⁢                        1                                                                                                  1                    +                                                                  ω                        I                        2                                            ⁢                                              τ                                                  c                          ⁢                                                                                                          ⁢                          1                                                2                                                                                                        )                                      ,                            Equation        ⁢                                  ⁢        4                                                      1                          T              1              SC                                =                                                    2                ⁢                                  S                  ⁡                                      (                                          S                      +                      1                                        )                                                              3                        ⁢                                          (                                  A                                      h                    _                                                  )                            2                        ⁢                          (                                                τ                                      e                    ⁢                                                                                  ⁢                    2                                                                    1                  +                                                            ω                      s                      2                                        ⁢                                          τ                                              e                        ⁢                                                                                                  ⁢                        2                                            2                                                                                  )                                      ,                            Equation        ⁢                                  ⁢        5            wherein [M] is the molar concentration of the paramagnetic ions, q is the number of coordinated water molecules per Gd, τm is the residence lifetime of the bound innersphere water molecule, and 1/T1M are the longitudinal proton relaxation rates. γl is the nuclear gyromagnetic ratio (γ(H)=42.6 MHz/Tesla), S is 7/2 for gadolinium ions, g is the electron g-factor, μB is the Bohr magneton, rGdH is the electron spin-proton distance, ωl and ωs are the nuclear and electron Larmor frequencies respectively, and A/h is the hyperfine or scalar coupling constant between the electron of the paramagnetic centre and the proton of the coordinated water. The correlation times are related by equations 6 and 7:
                                          1                          τ                              c                ⁢                                                                  ⁢                1                                              =                                    1                              τ                R                                      +                          1                              T                                  1                  ⁢                                                                          ⁢                  e                                                      +                          1                              τ                m                                                    ,                            Equation        ⁢                                  ⁢        6                                                      1                          τ                              e                ⁢                                                                  ⁢                1                                              =                                    1                              T                                  1                  ⁢                                                                          ⁢                  e                                                      +                          1                              τ                m                                                    ,                            Equation        ⁢                                  ⁢        7            wherein τR is the reorientational correlation time (or the rotational correlation time) and T1e is the longitudinal electron spin relaxation time of the metal ion.
The interplay of these parameters is shown in FIG. 1, and is complicated by the effect of magnetic field strength. The typical field strength of the clinical MRI scanners is 20-60 MHz (0.47-1.5 T). To present a clearer picture of the influencing parameters, the discussion of the observed longitudinal relaxation will be based on the 20 MHz field strength of clinical scanners. The optimization of the relaxivity can be achieved in various ways, the first of which is to increase the number of coordinated water q, which is directly proportional to the relaxivity as expressed by equation 1. Usually, q=2 is preferred for the nine coordinated gadolinium ion to optimize relaxivity and maintain a stable chelation. Recently, an acyclic ligand system was reported (Raymond et al., J. Med. Chem., 2005, ASPS Article) as having two innersphere coordinated water molecules, and was thermodynamically stable with relaxivity of 7.2-8.8 mM−1s−1. The second determinant that works together with q is the residence lifetime of coordinated water τm. There are two distinctive situations as shown in equations 3 to 7: one is a fast water exchange (T1M<<τm), in which the residence lifetime is the dominant factor, and another is a slow water exchange (T1M>>τm), where the relaxivity is dependent on the proton exchange and rotational and electronic relaxation time.
An increase in the water exchange rate (1/τm) increases the relaxivity and the optimal value of τm is 30 ns at 20 MHz. The major limitation of small molecular Gd-based clinical agents is the slow water exchange rate. A fast exchange rate is better associated with a slow rotational correlation time τR as the third influencing parameter in equation 6: that is, a slower tumbling rate of the molecule to enhance relaxivity. The formation of covalently or noncovalently bound macromolecules, such as dendrimers (Kobayashi et al., Bioconjugate chem., 2003, 14, 388; Sato et al., Magn. Reson. Med., 2001, 46, 1169 and U.S. Pat. App. Pub. No. 2004/0037777 A1), linear polymers (Aime et al., Inorg. Chem., 2000, 39, 5747) or proteins (Doucet et al., Invest Radio., 1990, 25, S53), can efficiently retard the rotational motion. Alternatives are micellar self-assembly (André et al., Chem. Eur. J., 1999, 5, 2977; Accardo et al., J. Am. Chem. Soc., 2004, 126, 3097; Anelli et al., Chem. Eur. J., 2001, 7, 5262) and incorporated liposomes (Bydder et al., Lancet, 1984, 3, 484; Nicolay et al., Bioconjugate Chem., 2004, 15, 799). However, the increase in relaxivity is less than expected due to internal flexibility, and the difficulty in obtaining a well-defined molecular dimension hampers further clinical application. Finally, a reduction in distance between the metal centre and the water protons rGdH increases the relaxivity, since relaxivity has a sixth-order dependence on the distance as expressed by equation 4. Estimation shows that a decrease of 0.2 Å leads to a 50% increase in the innersphere relaxivity (Raitsimring et al., Inorg. Chem., 2003, 42, 3972). By assessing different potential molecular architectures, the contrast agent development is accentuated to small molecular Gd-based contrast agents for higher relaxivity and specificity.
The first generation of clinically used contrast agents is listed in Table 1. The two embodiments of the Gd chelates are acyclic diethylenetriaminepentaacetic acid (DTPA) and cyclic 1,4,7,10-tetra(carboxymethyl)-1,4,7,10-tetrazacyclododecane (DOTA), the structures of which are shown in FIG. 2. They possess acetate pendant arms that wrap around the metal centre to form a stable gadolinium complex and provide hydrophilicity. Free gadolinium ions are highly toxic (Gd(III) aqua ion has LD50 of 0.1-0.2 mmol kg−1 in rats) and have to be strongly chelated by organic ligands to form stable complexes before administration.
TABLE 1HydrationChemical Name, GenericNumberRelaxivityName, Brand Name(q)Charge(mM−1 s−1)*Gd-DTPA, gadopentetate12−4.69dimeglumine, Magnevist ™Gd-BMA, gadodiamide,12−4.39Omniscan ™Gd-BMEA, gadoversetamide,12−4.70 (40)OptiMARK ™Gd-BOPTA, gadobenate12−5.20dimeglumine, MultiHance ™Gd-DOTA, gadoterate11−4.73meglumine, Dotarem ™Gd-HP-DO3A, gadoteridol,103.70 (40)ProHance ™Gd-DO3A-butrol, gadobutrol,103.60 (40)Gadovist ™*The relaxivities listed are at 20 MHz and 25° C., except those with the temperature in parentheses.
The clinical agents are regarded as intravenous extracellular agents with one innersphere water molecule, and the relaxivities fall in a range of 3-4 mM−1 s−1, which is far from the theoretical maximum of 100 mM−1 s31 1. They are distributed in the intravascular and interstitial space and are eliminated through the glomerular filtration, thus allowing the evaluation of physiological parameters. The bolus injection is adopted to increase the concentration to attain reasonably high signal intensity for these low relaxivity and non-specific agents. Although these agents are safe and are eliminated within 24 hours, high dosages of high osmolality agents may induce pain or increase the risk of in vivo dissociation, such as GD-DTPA with an osmolality of 1.96 osmol kg−1 versus that of 0.29 osmol kg−1 of body fluid. Their common disadvantages are poor efficiency (low relaxivity) and specificity (not tissue or organ targeting). Tissue-specific agents are in increasing demand for the detection of focal anomalies and evaluate tissue function for more accurate diagnosis. Research is undertaken to find a better contrast agent for the detection and characterization of focal liver pathology non-invasively, especially the hepatocellular carcinoma (HCC), thus notably reducing conspicuity in the diagnosis of hepatic diseases via the CT scan and biopsy.
A vast number of Gd-based contrast agents emerged following the use of Gd-DTPA in 0.5 M injections in early 1988. Polyaminocarboxylate ligands are widely included, and are composed of nitrogen and oxygen hard donor atoms, such as DTPA and DOTA, to securely chelate the gadolinium ion. Despite the introduction of liver-specific contrast agents such as the Mn-DPDP mangafodipir (the first small molecular agents) and SPIO ferumoxides (reticuloendothelial system specific agents) to the clinical realm, most agents are gadolinium based and have better safety profiles and well-controlled structures. The intravenous bolus injection of Gd-DTPA-BMEA or Gd-DTPA produces the transient enhancement of the liver and its vasculature in temporally distinct phases. In the phase III clinical trial reported in 1999, both agents showed improvement in lesion conspicuity, had good tolerance at the 0.1 mmol kg−1 dose, and had a well-documented record of excellent safety. These two gadolinium contrast agents depend on the hepatic arterial flow and intravenous bolus injection, which is not a sound contrast enhancing effect in liver imaging. Hence, significant hepatocellular uptake has to be achieved for further improvement.
The recently approved hepatocyte specific agent in the second generation of contrast agents is Gd-BOPTA (MultiHance™). It is an acyclic contrast agent that is based on DTPA with a lipophilic benzyl group, and was first reported by Vittadini et al. in 1988 (Vittadini et al., Invest. Radiol. 1988, 23, 246) and its clinical trials were completed in 2000. This monoaqua complex adopts a distorted tricapped trigonal prism with three nitrogen atoms and five carboxylate oxygen atoms that occupy eight coordination sites (Uggeri et al., 1995, 34, 633). The relaxivity 5.2 mM−1 s−1 of Gd-BOPTA is the highest among the four acyclic contrast agents. It distributes throughout the extracellular space with moderate clearance, followed by the hepatic uptake. The hepatic uptake is around 3-5% of the injected dose, and it displays a leveling off in the signal intensity upon a concentration increase, possibly by the saturation in the biliary excretion. These properties are postulated to be caused by the increase in hydrophobicity in the presence of the lipophilic side arm or an increase in intracellular microviscosity within the hepatocyte, combining interactions with transport proteins such as glutathione-S-transferase and non-specific intereactions with other proteins. The advantage is the standard dose of 0.1 mmol kg−1 can be reduced to 0.05 mmol kg−1 (Bracco, S.P.A., Italy, MultiHance™). Even with a reduced dose Gd-BOPTA has greater lesion to liver contrast during the delayed phase (40-120 min) of the contrast enhancement than Gd-DTPA. The tumor signal intensity can be improved because either the tumors do not have hepatocyte or reticuloendothelial systems, or the functioning of intratumoral hepatocytes is hampered. This agent is taken up by hepatocytes and is partially excreted via the biliary system, or is taken up by the Kupffer cells of the reticuloendothelial system (RES). Another hepatobiliary agent under clinical trial is Gd-EOB-DTPA, which has a specific uptake via hepatocytes but not hepatoma cells. These advances demonstrate the importance of specific uptake to a better quality of MRI imaging.
The liver efficiently extracts a large variety of albumin-bound amphipathic compounds from sinusoidal blood plasma. The Kupffer endothelial cell constitutes the sinusoidal endothelium, which is a barrier between the blood and the hepatocytes that can hamper the passage of viral particles to the hepatocytes. Hepatocytes have multispecific uptake systems, namely the sinusoidal (basolateral) and canalicular transport pathways. These include two hepatocellular carrier proteins, the Na+/taurocholate cotransporting polypeptides (NTCP) and the organic anion transporting polypeptides (OATP), which present at the basolateral plasma membrane of the hepatocyte (Meier et al., Hepatology, 1997, 26, 1667). As reported, the most probable carrier-mediated uptake pathway of hepatic contrast agents (Gd-BOTPA and Gd-EOB-DTPA) is through the OATP system and the possible biliary excretion pathways are excretion via ATP binding cassette (ABC) proteins, mainly by the multidrug resistance-associated protein 2 (mrp2) (Tiribelli et al., Biochemical and Biophysical Research Communications, 2001, 282, 60). The hydrophobic moieties enhance the uptake by the hepatocyte.
Acyclic ligands, such as Gd-DTPA and Gd-BOPTA, are subjected to kinetic lability. This endeavors to the investigation of cyclic ligands, such as Gd-DOTA, having selective and efficient complexation with their size-shape complementarity and preorganization. However, the formation of macrocycles is kinetically unfavourable. Much effort was devoted to the synthetic procedures for higher yield by either boosting the reactivity of the participating reagents and to suppress the linear oligomerization of reactants. In the regard of osmolality, neutral macrocyclic ligands are preferred. Under these two concerns, one class of macrocycle DTPA-bis(amide), which involves the cyclolization of DTPA dianhydride and diamine; yielding a convenient one-step and high yield preparation, attracts attentions. The three carboxylic arms encapsulate the gadolinium ion resulting in neutral complexes with the thermodynamic log KGdL of 15-19 (Meier et al., Hepatology 1997, 26, 1667).
Improvements in MRI contrast agents have been carried out in connection with: (1) relaxivity (e.g., >4 mM−1s−1) and in vivo stability, (2) specificity towards tissues or organs, (3) stabilities, and (4) percentage uptake by the hepatocytes (e.g., >5%).